Cellular therapy is a hopeful new approach to address unmet medical needs in patients. Currently, mesenchymal stem cells (MSCs) are used in multiple human clinical trials. However, it has become necessary to address problems such as differentiation regulation of mesenchymal stem cells for medical treatment.
Adipocytes and osteoblasts are differentiated from mesenchymal stem cells, and such differentiation is regulated by a transcription factor. The balance between adipogenesis and osteogenesis in mesenchymal stem cells is very important to repair/regenerate and maintain homeostasis. The disruption of controlling the balance of these processes during MSC differentiation leads to the disorders such as osteoarthritis and osteoporosis. PPARγ2 is expressed when mesenchymal stem cells are differentiated into adipocytes and involved in expression regulation of adipogenic genes. Also, Runx2 is expressed when osteoblasts are differentiated and involved in expression of osteogenic genes. Therefore, understanding the regulatory mechanism of transcription factors in osteoblast and adipocyte differentiation is very important.
PPARγ is a member of the PPAR family of transcription factors that includes PPARα, PPARγ, and PPARγ. PPARγ is a master regulator in adipogenesis, lipid biosynthesis, inflammation, and glucose metabolism. Alternative splicing produces PPARγ variants, including two major forms of the protein, PPARγ1 and PPARγ2. PPARγ2 differs from PPARγ1 by 30 additional amino acids on its N-terminus, and is expressed mainly in macrophages and adipogenic cells and partially expressed in bone marrow stromal cells. PPARγ1 is expressed in a wide range of tissues, including skeletal muscle, adipose tissue and bone. Binding of PPARγ to specific DNA sequences, including peroxisome proliferator-activated response element (PPRE) which consists of 2 direct repeats of the consensus nuclear receptor half-site separated by 1 base pair, requires heterodimerization with a second member of the nuclear receptor family, retinoic X receptor (RXR). This element is found in ap2 related to lipid storage and a CD36 promoter involved in cholesterol transport. The heterologous complex of PPARγ and RXR is associated with the nuclear receptor corepressor complex, including histone deacetylase (HDAC), nuclear receptor corepressor (NCoR) and silencing mediator for retinoid and thyroid receptors (SMRT) in the absence of PPAR ligand. Ligand binding to PPARγ triggers a conformational change and the corepressor complex is replaced by coactivators such as the p160/steroid receptor coactivator (p160/SRC) family, the mediator complex including PPARγ binding protein (PBP), PGC-1 (PPARγ coactivator-1) and CREB binding protein (CBP), and p300, leading to transcriptional initiation of target genes by a conformational change. Many transcription factors and ligands are involved in expression and function regulation of PPARγ. CCAAT/enhancer-binding protein (C/EBP) is directly bound to a PPARγ promoter to promote transcription. Prostaglandin J2, which is a natural PPARγ ligand, and thiazolidinediones (TZD), which is a synthetic reagent, for example, rosiglitazone and pioglitazone also increase a transcriptional activity of PPARγ. Retinoblastoma gene (RB) and cyclin D1 inhibit the transcriptional activity of PPARγ as a negative regulator.
Leucine zipper protein (LZIP) is a member of the large family of bZIP that belongs to the CREB/ATF gene family. LZIP includes a basic DNA-binding domain and a leucine-zipper domain that binds to a consensus cAMP-responsive element (CRE) and an AP-1 element. A human LZIP was identified as a host cell factor 1 (HCF-1) interacting protein that promotes cell proliferation and cellular transformation. N-terminal 92 amino acids of LZIP are a potent transactivation domain that consists of two LxxLL-transcriptional coactivator interaction motifs. LZIP includes five members, CREB3 (LZIP, Luman), CREB3L1 (OASIS), CREB3L2 (BBF2H7), CREB3L3 (CREB-H), and CREB3L4 (AIbZIP), which have a homology and different functions of transcription factors. Function of LZIP has been reported that LZIP binds to CCR1 and participates in regulation of Lkn-1-dependent cell migration. Also, LZIP binds to the CCR2 promoter, enhances expression of CCR2 and increases monocyte migration.
In recent years, a small LZIP, which is an isoform of LZIP, has been identified, and includes 354 amino acids having no transmembrane domain. sLZIP is not involved in LKN-1-dependent cell migration, activates HDACs, and thereby inhibits a transcriptional activity of a glucocorticoid receptor.
Accordingly, the inventors studied the regulation mechanism of sLZIP that regulates transcriptional activities of PPARγ and Runx2 in connection with differentiation of mesenchymal stem cells into osteoblasts and adipocytes, and thereby completed the invention.